The role of extrusion in controlling epithelial homeostasis Project Summary/Abstract Epithelia provide a protective barrier for the organs they encase; yet cells comprising this barrier are constantly renewed via cell death (apoptosis) and division. Surprisingly little is known about what triggers cells to naturally divide or die or how he number of cells dying and dividing are balanced during homeostasis. However, maintaining this balance is critical for maintaining barrier function and preventing diseases: if the death rate is higher, barrier function diseases such as asthma may result and if the division rate is higher, solid tumors will result. Through our investigations of how cells maintain a barrier when they die, we believe we have discovered a novel mechanism for how epithelia maintain homeostatic cell numbers, which we further investigate in this proposal. We previously discovered that when a cell dies within an epithelium, it signals its neighboring cells to form a contractile actomyosin rng to extrude it out. To do so, the dying cell emits the lipid Sphingosine 1-Phosphate (S1P), which activates extrusion by signaling the S1P receptor 2 (S1P2) in neighboring cells. More recently, we have found that in adult colon tissue and in developing zebrafish epidermis, epithelial cells appear to extrude prior to dying at sites of high cell crowding. Live cells also extrude in respons to experimental overcrowding using a chamber we devised. While both homeostatic and overcrowding-induced extrusion use the same S1P-S1P2 pathway we defined for apoptotic cell extrusion, the live cell extrusion pathway is activated by the stretch-activated channel Piezo 1. Importantly, we found that disrupting either stretch-activated signaling or the S1P-S1P2 pathway leads to cellular masses in both cell culture and developing zebrafish. Thus, we propose a model where the underlying tissue provides a space limit on epithelia so that any cell divisions and/or migrations cause overcrowding strain, which induces live cells to extrude. Extruding cells then die due to lack of survival signaling while promoting surrounding to survive and proliferate. We will test this hypothesis in Aims 1 and 2 by 1) investigating if extrusion promotes cell death in a cell culture model and 2) testing if cell masses in S1P2 zebrafish mutants result from lack of cell extrusion, increased proliferation, or both. To define what activates extrusion, we will 3) determine how Piezo 1 stretch-activated channels and calcium currents mediate S1P to trigger extrusion and 4) identify cell- cell or cell-matrix proteins critical for determining which cells extrude in response to uniform epithelial crowding forces. The proposed research is innovative because it investigates a novel mechanism for how epithelial cells maintain homeostatic cell numbers and uses multidisciplinary approaches, which include a zebrafish epidermal model we have developed to study epithelia and a cell overcrowding device we have developed with a bioengineer. The proposal is medically significant because misregulation of epithelial homeostasis is likely at the heart of most solid tumor formation and barrier function diseases.